Nanoscale coatings for encapsulation of biological entities

ABSTRACT

Methods, systems, and devices are disclosed for encapsulating biological entities with preservation of their biological activity. In one aspect, a method of encapsulating a biological entity includes templating a biocompatible material onto a biological structure to form a coating structure enclosing the biological structure, the coating structure having a size in the nanometer range, in which the coated biological structure preserves its biological activity within the coating structure. In some implementations of the method, the biological structure includes a virus and the biocompatible material includes silica.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent document claims benefit of priority of U.S. ProvisionalPatent Application No. 61/813,612, entitled “NANOSCALE COATINGS FORENCAPSULATION OF BIOLOGICAL ENTITIES” and filed on Apr. 18, 2013. Theentire content of the aforementioned patent application is incorporatedby reference as part of the disclosure of this patent document.

TECHNICAL FIELD

This patent document relates to nanoscale materials andnanotechnologies.

BACKGROUND

Nanotechnology provides techniques or processes for fabricatingstructures, devices, and systems with features at a molecular or atomicscale, e.g., structures in a range of one to hundreds of nanometers insome applications. For example, nano-scale devices can be configured tosizes similar to some large molecules, e.g., biomolecules such asenzymes. Nano-sized materials used to create a nanostructure,nanodevice, or a nanosystem can exhibit various unique properties, e.g.,including optical properties, that are not present in the same materialsat larger dimensions and such unique properties can be exploited for awide range of applications.

SUMMARY

Techniques, systems, devices, and materials are described forencapsulating biological entities with preservation of their biologicalactivity.

In one aspect, a method to produce a bioactive payload delivery deviceincludes forming an intermediate structure by binding a polymer materialwith a biological substance based on an electrostatic force, in whichthe formed intermediate structure includes a plurality of regionspresenting a net surface charge, and forming a coating structure of abiocompatible material directly on the formed intermediate structure toenclose the biological substance, in which the coating structurepreserves biological activity of the biological substance enclosedtherein, thereby producing a bioactive payload delivery device.

In another aspect, a bioactive payload delivery device includes aninterior material structure including a polymer material and abiological substance that are bound to each other via an electrostaticinteraction, in which the interior material structure includes aplurality of regions presenting a net surface charge, and an exteriornanostructure formed of a biocompatible material to encapsulate theinterior structured material, thus preserving biological activity of theencapsulated biological substance.

In another aspect, a method for encapsulating a biological substanceincludes forming a biocompatible material onto a biological structure toform a coating structure enclosing the biological structure, the coatingstructure having a size in the nanometer range, in which the biologicalstructure preserves biological activity within the coating structure. Insome implementations of the method, the biological structure includes avirus and the biocompatible material includes silica.

The subject matter described in this patent document can be implementedin specific ways that provide one or more of the following features. Thedisclosed methods can be implemented to encapsulate biological entitiesor other substances within a synthetic matrix, e.g., such as silica in ananoparticle format, where the encapsulated bioentities are without anyloss or with minimal loss of their activity. The synthesizednanostructured matrix (e.g., silica nanoparticle enclosure) provides theability to target the release of the biologicals, e.g., through anexternally triggered mechanism and/or incorporation of targetingmoieties to the synthesized nanostructured matrix. In someimplementations, the method can include effectively changing the surfacecharge on the biological entity through cross reaction with a cationicpolymer like poly-L-lysine. The method can include a charge mediatedsilica sol-gel condensation reaction directly onto the surface of thebiological entities, e.g., forming an enveloping silica matrix coatingaround the biologicals giving rise to a nanoparticle. In someimplementations, for example, a sensitizing agent such as fluorocarbonemulsions can be used as ultrasound triggered cavitation centers and beco-encapsulated with the biological entities, e.g., bound to thebiological entity prior to the exemplary silica sol-gel condensationreaction to allow for an externally triggered release mechanism to bebuilt into the nanoparticle. For example, once the nanoparticle isformed, additional surface modifications can be made to the nanoparticleusing various chemistries including silane chemistry to add a variety offunctional characteristics to the nanoparticle, including, but notlimited to, polyethylene glycol (PEG) for immune response evasion,targeting moieties for specific delivery or release, environmentalsensing moieties (e.g., redox, hypoxic, acidic, etc.) for targetedrelease. Since the exemplary synthetic nanostructured matrix preventsthe encapsulated biological entities from being directly exposed toserum and tissue conditions prior to being released, the encapsulatedbiological entities are protected from degrading conditions present inserum, e.g., allowing for an enhanced activity half-life in vivo. Also,for example, the ability to selectively trigger the release of thebiologicals results in lower systemic toxicity giving rise to a bettertolerated therapy regime.

For example, the disclosed biocompatible coating nanostructure can beeasily functionalized and utilized to encapsulate biological entitiessuch as viruses (e.g., adenoviruses). Exemplary implementations usingscanning electron microscopy (SEM) analysis to characterize exemplarycoating nanostructures shows discrete mono-disperse particles withnanoscale features. Exemplary implementations also showed that exemplarynanostructure-coated viruses retained transduction ability and wereprotected from proteinase k and neutralizing antibodies. Additionally,for example, implementations of the disclosed technology showed theability to ablate uptake of the exemplary coated virus byfunctionalizing the exemplary coating with polyethylene glycol (PEG).For example, subsequent conjugation of cRGD to the exemplary coatingrescued the transduction ability of the viruses. The exemplary coatingis robust, and the coated virus can be stored at −80° C. without loss inactivity. Applications of the disclosed technology can include, but arenot limited to, biotechnology and biomedicine, e.g., such as in thetreatment and monitoring of numerous diseases including cancer,diabetes, among others. For example, the disclosed technology can beused as a therapeutic approach for clinical translation with the goal ofopening up the use of oncolytic viruses to more cancer therapeuticapplications and improving the clinical response rates of oncolyticviruses. Furthermore, the disclosed nanocoating platform may enableadditional gene therapy based approaches targeted at cancer metabolismand other genetic based diseases.

Those and other features are described in greater detail in thedrawings, the description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows an illustrative diagram of the exemplary method for ananoscale template driven encapsulation process of biological entitiesthat preserves biological activity.

FIG. 1B shows a scanning electron microscopy (SEM) image of an exemplarysilica nanostructure coating of a virus.

FIG. 2A-2C show data plots and images from exemplary implementationsusing the exemplary silica nanostructure coating encapsulating viruses.

FIG. 3 shows a process diagram of an exemplary method forco-encapsulation of biological entities with targeting moieties in thenanostructure enclosure.

FIG. 4 shows data plots of an exemplary neutralization assay in thepresence of antibodies.

FIG. 5 shows a data plot of exemplary implementations for retargeting ofthe exemplary silica-coated biological entity.

FIG. 6 shows data plots depicting ultrasound triggered release ofadenoviruses from the exemplary nanostructured coatings.

DETAILED DESCRIPTION

Oncolytic viral therapy is a cancer therapy that uses viruses toselectively replicate in and kill tumor cells. Over the last twodecades, significant progress has been made in the preclinical andclinical development of viral-based therapy as a platform for thetreatment of cancer. While much progress has since been made inunderstanding viral lifecycles and biology, their delivery and clearancecharacteristics remain a major stumbling block for effective therapy.For example, some patients mount immune response to the viral therapy,e.g., which can cause and increase side effects in treatment of thepatient, and overall, limits the clinical efficacy of this therapeuticapproach. Current methods to address the challenges of viral therapytechniques such as immune response focuses on direct modification ofvirus, or encapsulation. Also, harsh chemical reaction conditions duringtreatment also impact viability of the viral therapy. For example,efficacy of viral therapy also depends on natural affinity of viralvectors for target cells (e.g., host cell tropism). It is believed thatthe ability to effectively deliver therapeutic viruses systemically needtechnology to address these challenges and thereby expand the efficacyof the oncolytic viral platform to patients with disseminated disease.

Techniques, systems, devices, and materials are described forencapsulating biological entities using nanoscale material designs andspecialized functionalization. Implementations of the disclosedtechnology include biocompatible nanocoatings that preserve thebioactivity of a viral payload and protect the viral payload from immunerecognition and neutralization during viral therapy treatment, withspecific targeting, improved circulation, and theranostic abilities.

In some aspects, the disclosed technology includes a nanoscale templatefabrication process to encapsulate a biological entity (e.g., such as avirus) in an nanostructured enclosure (e.g., such as a nanoparticlecoating structure). In addition to encapsulating the biological entity,the nanostructured enclosure also has the ability to target the releaseof the biological entity through an external trigger and/or theincorporation of targeting moieties in the nanostructure enclosure. Forexample, the disclosed technology can be applied in a variety of fieldsin biomedicine and biotechnology, e.g., including viral therapies.

In some implementations, a viral vector can be encapsulated in asilica-based nanoparticle coating structured to provide a controlledrelease mechanism to target the release of the virus payload, in vitroand in vivo, using an external trigger and/or incorporated targetmoieties. In some examples, an external acoustic signal can be appliedto the exemplary nanostructured coating containing the viral vector tocause release of the viral vector at a target site within an organism,e.g., such as at a tumor within an animal.

Exemplary implementations using adenoviruses encapsulated by theexemplary silica nanocoating structures are described, e.g., conferringprotection against proteinase K. For example, results of the exemplaryimplementations as shown herein demonstrated activation using ultrasoundof viral transfection in a human pancreatic cell line. In otherimplementations, for example, the disclosed encapsulation approach canbe adopted for materials other than silica, e.g., including, but notlimited to, titanium oxide and calcium phosphate.

In some implementations, for example, the exemplary silica-basednanoparticle coating can be formed by the direct condensation of aremovable silica matrix on the surface of biological entities to formthe nanoparticle while preserving biological activity. Using thedisclosed fabrication techniques, for example, the exemplary silicamatrix can be formed directly on the surface of the biological entitiesunder biologically compatible reaction conditions. This allows higherencapsulation efficiency without consequent loss of activity of thebiological entities. This also brings fine control over particle sizegiving nanoparticles with well-defined size characteristics. Forexample, silica can be used to form the nanostructure coating, e.g.,particularly in biological applications due to its biocompatibility andbiodegradability. Additionally, for example, the disclosed technologycan include a sensitizing agent within the synthesized structure toallow externally triggered release of the encapsulated biologicalentities, as well as exemplary methods for the functionalization of thenanoparticle for exemplary characteristics like improved circulationtime and targeting benefiting from easy functionalization of silicasurface.

In some aspects, the disclosed technology includes a method ofencapsulating biological entities and biological substances within asynthetic matrix, e.g., such as silica in a nanoparticle format, withoutany loss or with minimal loss of activity of the biological entities orsubstances. For example, the produced synthetic matrix enclosure (e.g.,nanoparticle) encapsulating the biological entities or substances can beconfigured to have the ability to selectively release the biologicalentities or substances through an externally triggered mechanism and/orthe incorporation of targeting moieties to the synthetic matrixenclosure. For example, the exemplary method can include a process fortemplating silica onto biological entities, e.g., viruses, whilepreserving biological activity/compatible with life. In someimplementations, the method can include a process for changing thesurface charge on the biological entities, e.g., through cross reactionwith a poly-cationic substrate (e.g., such as poly-l-lysine) to apositively charged surface compatible with the silica polycondensationprocess. In some implementations, the method can include a process toimplement a charge mediated silica sol-gel condensation reactiondirectly onto the surface of the biological entities or substancesforming an enveloping silica matrix coating around the biologicalentities or substances giving rise to a nanoparticle. For example, asensitizing agent such as fluorocarbon emulsions as ultrasound triggeredcavitation centers can be included prior to the silica sol-gelcondensation reaction to allow for an externally triggered releasemechanism to be built into the particle. In some implementations, themethod can include a process to encapsulate a drug or a therapeuticagent with the biological entity. In some implementations, the methodcan include a process to include iron oxide or other materials (of anano-particulate nature) into the produced silica coating, e.g., forimaging. Once the nanoparticle is formed, for example, the method caninclude implementing additional surface modifications to thenanoparticle using various chemistries, e.g., including silane chemistryto add a variety of functional characteristics to the nanoparticle,e.g., such as PEG for immune evasion, targeting moieties for specificdelivery or release, environmental sensing moieties (e.g., redox,hypoxic, acidic, etc.) for targeted release, among others. For example,since the biologicals are not being directly exposed to serum and tissueconditions prior to being released, the encapsulated biologicals areprotected from degrading conditions present in serum allowing for anenhanced activity half-life in vivo. For example, the ability toselectively trigger the release of the biologicals results in lowersystemic toxicity giving rise to a better tolerated therapy regime. Insome implementations, the method can include stabilizing theencapsulated biological entity allowing for improved thermal stabilityat −80° C. to −37° C. For example, lyophilization or critical pointdrying of the nanoparticle can be performed while preserving thebiological activity of the encapsulated entity.

FIG. 1A shows an illustrative diagram of the exemplary method for ananoscale template driven encapsulation process of biological entitiesthat preserves the biological activity of the encapsulated biologicalentities. The method includes a process to form an intermediarybiomaterial structure 105 by binding a surface-charged material 101 witha biological entity or substance 102 such that the formed intermediarybiomaterial structure 105 presents regions having a net surface charge,e.g., which may be modified from that of the biological entity substance102. For example, as illustrated in the diagram of FIG. 1A, a negativelycharged virus (e.g., adenovirus) is reacted with a cationic polymer,poly-L-lysine (PLL), to modify the surface charge on the adenovirus,e.g., in which the PLL is bound to the surface of the adenovirus by anelectrostatic force. The method includes a process to form biologicalentity-encapsulated nanostructure 110 by forming a nanostructure coating109 to enclose the intermediary biomaterial structure 105, in which theencapsulated bioentity or substance 102 maintains its bioactivityfunctionality. For example, as shown in FIG. 1A, the exemplarypositively charged viral-material structure attracts negatively chargedsilica precursor and hydroxyl ions creating a basic environment suitablefor a silica polycondensation reaction to form the nanostructure coating(e.g., silica-based nanoparticle) that encapsulates the viral payload.FIG. 1B shows an SEM image of an exemplary silica nanocoating of suchviruses (e.g., referred to as a siVirus platform).

For example, silane chemistry is typically compatible with physiologicalconditions. The disclosed methods can manipulate silane chemistry toachieve coating of viruses into silica nanoparticles without anyconsequent loss of biological activity of the viruses, as shown later inFIGS. 2A-2C. Traditional sol gel synthesis methods use alkaline pH, acidcatalysis or addition of salts. The subsequent gelation occurs in bulkin the liquid phase with downstream processing and aging of the gel toform silica compositions. Using the described fabrication methods, thesol gel reaction occurs at neutral pH and becomes template driven withthe nanomaterial matrix (e.g., exemplary silica matrix) being formeddirectly on the surface of the biological entity to be encapsulatedunder biologically compatible reaction conditions.

In some implementations, for example, a charged template is used as aninitial starting point of the reaction. Depending on the initial surfacecharge of the template, the template is re-functionalized to carry a netor partial positive charge via electrostatic interaction with apoly-cationic polymer like poly-L-lysine, e.g., thereby forming abiotemplate-material structure. Silicic acid is then added to thereaction mixture in a concentration where nucleation of bulk gelation ofsilica from sol occurs slowly. Absorption of silicic acid to the surfaceof the template occurs. When local concentration of silicic acid ions atthe surface of the template surpasses the threshold for nucleation ofsilica gel, a self-limiting poly-condensation occurs on the surface ofthe template encasing the template in a matrix of silica gel. Forexample, the initial template can be accompanied by additionalfunctional moieties for co-encapsulation. The reaction occurs atphysiological conditions and allows higher encapsulation efficiencywithout consequent loss of activity of the biologicals. This also bringsfine control over particle size giving nanoparticles with well-definedsize characteristics. Additionally, for example, the silica coating caneasily accept secondary functionalizations to achieve a broad range ofmaterial properties.

The exemplary method can include a wet-chemical fabrication processusing silica sol-gel chemistry that uses a colloidal solution (sol) as aprecursor for an integrated network (gel) of discrete particles ornetwork of amorphous silica, e.g., such as:

Si(OCH₃)₄+2H₂O→SiO₂+4CH₃OH.

Examples of possible functional moieties for added functions include,but are not limited to, nano-emulsions for ultrasound activation,chemotherapy drugs like doxorubicin, standard therapeutic drugs likestatins, therapeutic compounds like small molecule inhibitors, DNAvectors, RNA vectors for shRNA and RNAi, microRNA, and MRI contrastagents like gadolinium and radio-contrast dyes.

Examples of secondary functionalization include, but are not limited to,PEG, pH sensitive PEG, affibodies, antibodies, IgG, IgM, targetingligands like VEGF-C, cRGD and folate, DNA and aptamers, proteins,lipoproteins, apolipoproteins, glycoproteins, glycans, carbohydrates,saccharides and oligosaccharides, polymers and oligomers, and lipids.

The disclosed template-driven nanoencapsulation technology preservesbiological activity of proteins, enzymes, DNA vectors, viruses, bacteriaand other sensitive biological agents that would otherwise not be ableto maintain biological activity in vivo to provide a degree ofprotection to the encapsulated agent. In some implementations, thepresent technology includes a template-driven silica polycondensationprocess to produce well defined particles with controllable sizecharacteristics in the nanoscale regime. For example, thetemplate-driven silica polycondensation process includes directlytemplating silica on the agent to be encapsulated, which can enableencapsulation of a broad range of agents without size constraints withvery high efficiencies. The disclosed nanomaterial structures can beproduced and utilized as (1) a biocompatible coating that allows forimmune system evasion and protection from degradation; and (2) ascaffold to incorporate additional functional moieties, e.g., toretarget the virus, decrease biochemical affinity to inhibit adhesionand uptake (e.g., PEGylation, charge, zwitterionic), increasebiochemical affinity with targeting ligands (e.g., mAb), or enablingultrasound-triggering (e.g., perfluorocarbon nanoparticle, cavitationnucleation site).

In some exemplary implementations of the disclosed technology, viruseswere encapsulated in silica nanocoating structures.

Viruses are highly evolved systems with gene transfer and lyticmechanisms that can be used for medicinal applications. However, therecan be some technical issues to be addressed in such applications. Forexample, the immune system may be a barrier to these approaches, throughits ability to recognize these viruses, neutralize them, and/oraccelerate their clearance and degradation. Also, for example, thespecific host cell tropism of viruses limits their application tospecific cell populations. In this context of a viruses and otherpathogens affecting a host tropism or cell tropism, tropism refers tothe way in which different viruses/pathogens have evolved topreferentially target specific host species or cell types within aspecies.

Using oncolytic viruses as an example, oncolytic viruses can selectivelyreplicate in and kill tumor cells, and over the last two decadessignificant progress has been made in the preclinical and clinicaldevelopment of viral based therapy as a platform for the treatment ofcancer. For example, the use of Onyx-015 demonstrated the safety of thisapproach, and significant responses were noted among patients treated byintralesional injections and regional vascular delivery. However,induction of high titers of neutralizing antibodies as well as highlevels of antiviral cytokines was demonstrated among the cancer patientstreated with Onyx-015, limiting the efficacy of this approach bysystemic delivery. Despite these limitations, intratumoral and regionalapproaches are being pursued in large, multinational studies withgrowing evidence of success in melanoma and hepatocellular carcinoma.While much progress has been made in understanding viral lifecycles andbiology, their delivery and clearance characteristics remain a majorstumbling block for effective therapy. The human body is exceedinglyeffective in clearing most pathogens from circulation. While it maytolerate an initial dose of viruses for therapy, follow up doses arerapidly cleared with little therapeutic benefit. Additionally the issueof host cell tropism limits the utility of viruses only to cells withwhich they have affinities with. Addressing the issues of immuneactivation and host cell tropism would be key to advancing viraltherapy. Implementations of the disclosed technology provides theability to effectively deliver therapeutic viruses systemically andexpand the efficacy of an oncolytic viral platform to patients withdisseminated disease.

The issue of immune activation following viral therapy has beenchallenging to overcome. For example, it has been shown in animal modelsthat prior exposure to a given virus limits therapeutic outcome in mice.Clinical trials of viral therapy in humans have noted the induction ofhigh titers of neutralizing antibodies and elevated cytokine levels,which would limit the possibilities for their use in vivo. For example,viruses currently in clinical trials are derivative of common humanpathogens that humans are exposed to on a regular basis. Prior exposureto these viruses complicates interpretation of the clinical outcome andnegates the possibility of repeat therapy to clear residual disease. Asecondary concern arising from immune activation is the shortpersistence time in blood and bioavailability of therapeutic viruses.The speed at which viruses are cleared from the immune system precludesgeneral systemic delivery of therapeutic viruses. Most conventionalapproaches (e.g., in clinical trials) are focused on intratumoral,intralesional or intra-arterial delivery of viruses for regionaltherapy. The inability to achieve systemic delivery precludes theability to properly treat disseminated and systemic diseases. Thelimitations on repeat dosing and lack of a true systemic therapy optionfrom immune activation is a first and paramount hurdle that needs to becrossed for greater clinical uptake of viral therapy.

The issue of host cell tropism is particularly problematic because itimplicates three areas of therapy, for example, the therapeutic targetsthat can be treated, variable receptor expression between patients, andoff target toxicities. Just as flu virus that normally infects lungtissues would have difficulty gaining traction in the prostate orpancreas, individuals within a given population have differentsusceptibilities to different viral systems. Receptors that viruses useto gain entry into a cell have highly variable expression levels incancer cells, which can lead to off target toxicities in tissues withhigh expression levels of these receptors (splenic and liver toxicitiesare often implicated).

The disclosed technology addresses the issue of immune activation and isable to retarget viruses to cell markers that are over expressed indisease conditions and expand the efficacy of the viral platform.Moreover, the disclosed technology opens up the use of viruses to moretherapeutic applications to improve the clinical response rates.Furthermore, the disclosed siVirus platform enables additional genetherapy based approaches targeted at cancer metabolism and other geneticbased diseases.

Changing the interaction surface a virus presents to the body impactsthe type of response it elicits from downstream processes such as immunesystem responses and cellular uptake. In some embodiments of thedisclosed nanocoating technology, silica is used, as it lends itselfwell as a coating material for viruses due to its favorablebiocompatibility, degradation characteristics and flexible silanechemistry which allows for simple modification of the silica coating.Exemplary implementations using the exemplary silica nanocoatingstructures indicate that coating adenoviruses with silica improved theirtransduction ability with the silica layer rapidly degrading underendosomal conditions. Silica-coated viruses are also protected fromneutralizing antibodies and proteinase K.

The disclosed technology can be used to change the interaction surface avirus presents to the body impacts the type of response it elicits fromdownstream processes like immune system responses and cellular uptake.In exemplary implementations performed and described herein, the abilityto incorporate PEG and peptide components to ablate cellular uptake andachieve receptor mediated uptake respectively is demonstrated. Forexample, the exemplary silica coating is robust and the coated virus canbe stored at −80° C. without loss in activity.

FIG. 2A shows a data plot depicting the transduction efficiency of anexemplary Adenovirus-RFP bioagent payload after coating with anexemplary silica nanostructure. RFP fluorescence intensity was measuredtwo days post transduction with setups normalized to the MOI 500 level.The exemplary silica-coated adenoviruses maintained transductionactivity and were protected from proteinase K digest. An exemplarysecondary coating of PEG on the silica particle was sufficient to ablatetransduction.

FIG. 2B shows a data plot depicting the FACS analysis (e.g.,fluorescence-activated cell sorting, using flow cytometry) of cellstransduced with free adenovirus-RFP or silica-coated adenovirus-RFP.Cells were infected with Ad-RFP then harvested and analyzed on FACS twodays post transduction. Both free and silica-coated Ad-RFP showedpopulation wide dose response with increasing viral titer.

FIG. 2C shows a data image showing in vivo activity of the exemplarysilica-coated adenoviruses. An exemplary C57/Bl6 nu/nu mouse bearingbilateral HT1080 tumors was given two injections of 5×10⁷ pfu freefirefly luciferase encoding adenoviruses (Ad-Luc) intratumoral (IT) tothe right tumor and two doses of 5×10⁷ pfu silica-coated Ad-Luc IT tothe left tumor. Luciferase expression was analyzed after 4 days postinjection by giving 50 μL 0.2 mg/mL d-luciferin IV.

The disclosed technology is also capable to co-encapsulate variousmoieties that confer added functionality with our viruses. For example,a perfluorocarbon nanoemulsion can be included to allow for ultrasoundtriggered release of the virus. In one such exemplary implementation ofthe method to co-encapsulate an exemplary moiety, a fluorocarbonemulsion is stabilized by a negatively charged surfactant. The emulsionis reacted with a cationic polymer, e.g., such as poly-L-lysine (PLL),followed by adsorption of negatively charged viruses. Finally, silicapoly-condensation reaction is performed on the surface encapsulatingemulsion together with viruses in silica.

FIG. 3 shows a process diagram of an exemplary method forco-encapsulation of one or more biological entities with targetingmoieties in the nanostructure enclosure, in which the nanostructureenclosure preserves the biological activity of the encapsulatedbiological entities while providing a targeting release mechanism. Themethod includes a process to bind a surface-charged material 301 with atargeting substance or moiety 302 to form a material complex 303presenting surface charge. For example, a negatively chargednanoemulsion (NE) can be reacted with a cationic polymer, e.g.,poly-L-lysine (PLL), to form a material substance having positivelycharged regions on the substance containing the PLL and the NE based onthe PLL. The method includes a process to form a bioentity-targetingmaterial structure 307 by reacting the surface-charged material complex303 with a biological entity or substance 305, e.g., which binds thebiological entity or substance to the material complex 303 on thesurface charge. For example, as illustrated in the diagram of FIG. 3, anegatively charged virus (e.g., adenovirus) can be reacted with thecationic polymer, PLL, regions of the structure 307, e.g., in which theadenovirus is bound to the surface of the PLL-NE structure by anelectrostatic force. The positively charged PLL-NE attracts negativelycharged adenoviruses to its surface. Silica poly-condensation reactionis performed on the surface encapsulating emulsion together with virusesin silica. The method includes a process to produce a biologicalentity/targeting substance co-encapsulated nanostructure 310 by forminga nanostructure coating 309 to enclose the structure 307. For example,the exemplary positively charged virus/PLL-NE structure attractsnegatively charged silica precursor and hydroxyl ions creating a basicenvironment suitable for the silica polycondensation reaction to form asilica nanocoating that encapsulates the viral payload with thetargeting substance, e.g., the nanoemulsion.

In some exemplary implementations of the disclosed technology, virusesand targeting moieties were encapsulated in silica nanocoatingstructures.

All adenoviruses used in the exemplary implementations described belowwere non-replicative with deletions in the E1/E3 regions. To determineif the exemplary silica nanocoating had an adverse effect on thetransduction efficacy of adenoviruses, free-versus silica-coatedRFP-encoding adenovirus (Ad-RFP) were compared in cell culture. Theexemplary results showed that silica-coated adenoviruses not onlymaintained activity post coating but also showed a 3-fold increase intransduction efficacy over free adenoviruses, e.g., which could be anearly indication of a retargeting mechanism. Subsequent implementationsusing FACS analysis, for example, indicated this was a population widephenomena and not the result of a small secondary population ofsuper-infected cells. Furthermore, the exemplary implementationsincluded the addition of PEG to the silica layer, which was shown toablate transduction by the silica-coated adenoviruses. The exemplaryadenoviruses encapsulated in the exemplary silica nanocoatings can alsobe stored at −80° C. without any lost in bioactivity. Exemplary in vivoimplementations in mice showed the 3-fold higher activity from coatedviruses carried over also applied in vivo.

Exemplary implementations were performed to demonstrate the protectionof the silica-coated adenoviruses from neutralizing antibodies. Thetransduction efficacy of free and coated adenoviruses were compared postexposure to goat Ad5-anti-hexon polyclonal (e.g., obtained from ThermoScientific PA128357) for 1 hour at 37° C. Free adenovirus showed a sharpneutralization with a inhibitory concentration 50% (IC50) of 5×10⁻³antibody dilution. The exemplary silica-coated adenovirus showed no lossof activity at the same antibody concentration.

FIG. 4 shows data plots depicting exemplary results of neutralizationassays, in the presence of antibodies, to characterize the bioactivitypreservation of the exemplary bioentity-encapsulated nanostructures. Forexample, free- and silica-coated Ad-RFP were incubated with varyingdilutions of neutralizing antibodies. RFP fluorescence intensity wasmeasured two days post transduction with setups normalized to the MOI500 level. Transduction with free adenoviruses was significantlysuppressed in the presence of neutralizing antibodies at the twoexemplary antibody titers tested. The exemplary silica-nanocoatedadenoviruses showed little differential impact from incubation withneutralizing antibodies, with only a modest suppression at the higherantibody titer.

Exemplary implementations were performed to demonstrate the ability toretarget adenoviruses using targeting ligands conjugated to the surfaceof the exemplary silica nanostructured coating. For example, exemplarysilica-nanocoated Ad-RFP was used and given a secondary coating of PEGto ablate transduction. For example, Ad-RFP was coated with silica andPEG, and alternatively functionalized with cRGD or β-mercaptanol. RFPfluorescence intensity was measured 2 days post transduction with setupsnormalized to the MOI 500 level. FIG. 5 shows a data plot showing cRGDretargeting of silica-nanocoated Ad-RFP. The exemplary results of theimplementations showed that functionalization with cRGD was sufficientto rescue PEG/silica-coated Ad-RFP from PEG ablation. β-mercaptanolfunctionalized PEG/silica Ad-RFP showed no recovery in transductionefficiency.

Exemplary implementations were performed to demonstrate the ability totrigger viral release using ultrasound exposure. For example, silicananocoating structures that encapsulated adenovirus (siAd-RFP) werecompared to silica nanocoating structures that co-encapsulatedadenovirus with a nanoemulsion (siAd-RFP-NE). FIG. 6 shows data plotsdepicting ultrasound triggered release of adenoviruses. The exemplarysiAd-RFP and siAd-RFP-NE were alternatively exposed to ultrasound orleft as-is. The exemplary siAd-RFP-NE showed a 20 fold increase in RFPtransduction post exposure to ultrasound. The exemplary siAd-RFP showedno significant difference in RFP transduction post ultrasound exposure.

Examples

Some exemplary embodiments of the methods and devices of the presenttechnology are described below.

In one example, a method to produce a bioactive payload delivery deviceincludes forming an intermediate structure by binding a polymer materialwith a biological substance based on an electrostatic force, in whichthe formed intermediate structure includes a plurality of regionspresenting a net surface charge; and forming a coating structure of abiocompatible material directly on the formed intermediate structure toenclose the biological substance, in which the coating structurepreserves biological activity of the biological substance enclosedtherein, thereby producing a bioactive payload delivery device.

Implementations of the exemplary method can include one or more of thefollowing exemplary features. For example, in some implementations ofthe method, the coating structure can include a nanoparticle having asize in the nanometer regime, e.g., such as 1 nm to 999 nm. For example,the biological substance can include a virus, bacteria, protein, enzyme,prodrug (e.g., a drug precursor molecule that can be converted into amore reactive form by some physical or chemical means), and/or nucleicacid vector, e.g., such as a DNA vector or an RNA vector. For example,the biocompatible material used to form the coating structure caninclude silica. In some implementations of the method, for example, theforming the intermediate structure can include cross-reacting thebiological substance with a poly-cationic polymer material to form apositively-charged surface in the plurality of regions of theintermediate structure. For example, the poly-cationic polymer materialcan include poly-l-lysine. In some implementations of the method, forexample, the forming the coating structure can include a charge-mediatedsilica sol-gel condensation reaction directly onto the surface of theintermediate structure, in which the formed coating structure includesan enveloping silica matrix encapsulating the biological substance. Insome implementations, for example, the method can further include, priorto the forming the coating structure, binding a targeting moiety to theintermediate structure to enable controlled release of the biologicalsubstance from the coating structure. For example, in someimplementations, the method can further include deploying the producedbioactive payload delivery device in a fluidic media to a targetsubstance; and applying an external triggering signal to an areaincluding or proximate to the target substance to cause the targetingmoiety to release the biological substance from the coating structure tothe target substance.

Implementations of the exemplary method can include one or more of thefollowing exemplary features. In some implementations, for example, themethod can include adding a sensitizing agent to produce ultrasoundtriggered cavitation centers inside the coating structure, in which theadding the sensitizing agent is implemented prior to the forming thecoating structure. For example, the sensitizing agent can include afluorocarbon nanoemulsion. For example, the method can further includedelivering the biological substance to a target cell or tissue in aliving organism, in which the delivering can include injecting theproduced bioactive payload delivery device through vasculature of thebody, e.g., in which the bioactive payload delivery device extravasatesfrom the vasculature to the target cell or tissue; and applying acousticenergy (e.g., an ultrasound pulse(s)) to an area of the living organismincluding or proximate to the target cell or tissue to cause thesensitizing agent to rupture the coating structure and release thebiological substance to the target cell or tissue.

Implementations of the exemplary method can include one or more of thefollowing exemplary features. In some implementations, for example, themethod can include adding a pharmaceutical drug or a therapeutic agentwith the intermediate structure to be enclosed in the coating structure,in which the adding the pharmaceutical drug or the therapeutic agent isimplemented prior to the forming the coating structure. In someimplementations, for example, the method can include adding an ironoxide constituent or other nanoscale material with the biocompatiblematerial to form the coating structure, in which the added iron oxideconstituent or the other nanoscale material provides an agent to enhanceimaging of the coating structure. For example, the coating structure canstabilize the biological substance enclosed therein, thereby allowingthermal stability at temperatures including 80° C. to −37° C. In someimplementations, for example, the method can include lyophilizing orcritical point drying the produced bioactive payload delivery device, inwhich the coating structure preserves biological activity of thebiological substance enclosed within the lyophilized or critical pointdried bioactive payload delivery device.

Implementations of the exemplary method can include one or more of thefollowing exemplary features. In some implementations, for example, themethod can include functionalizing an exterior surface of the coatingstructure. For example, in some implementations, the functionalizing caninclude adding polyethylene glycol (PEG) to provide a secondary coatingcapable of preventing an immune system response within a living organismin which the bioactive payload delivery device is deployed. For example,in some implementations, the functionalizing can include attaching atargeting ligand capable of selectively binding to a particular regionof a cell or tissue of a living organism in which the bioactive payloaddelivery device is deployed. For example, in some implementations, thefunctionalizing can include attaching an environmental sensing moiety tothe external surface, the environmental sensing moiety capable ofchemically changing form based on at least one of a redox reaction,hypoxic reaction, or acidic pH in a local environment to which thebioactive payload delivery device is deployed.

In one example, a bioactive payload delivery device includes an interiormaterial structure including a polymer material and a biologicalsubstance that are bound to each other via an electrostatic interaction,in which the interior material structure includes a plurality of regionspresenting a net surface charge, and an exterior nanostructure formed ofa biocompatible material to encapsulate the interior structuredmaterial, thus preserving biological activity of the encapsulatedbiological substance.

Implementations of the exemplary device can include one or more of thefollowing exemplary features. For example, the exterior nanostructure ofthe device can protect the biological substance from degradation fromexternal environmental factors, e.g., including pH, temperature,pressure, and chemical substances, in an environment where the bioactivepayload delivery device may be deployed. For example, the outer surfaceof the exterior nanostructure of the device can be functionalized with atumor targeting ligand to cause the bioactive payload delivery device toselectively accumulate in a particular tumor region over other tissues.For example, the outer surface of the exterior nanostructure of thedevice can be functionalized with an agent to increase circulation timeby reducing uptake from undesired body tissues, organs, and systems,e.g., in which the agent includes polyethylene glycol, a zwitterioniccompound, and/or a patient-specific coating such as cell membranes. Forexample, the biological substance can include a virus, bacteria,protein, enzyme, prodrug, or nucleic acid vector (e.g., such as a DNAvector or an RNA vector). For example, the biocompatible material caninclude silica.

Implementations of the exemplary device can include one or more of thefollowing exemplary features. In some implementations, for example, thedevice further can include an acoustic sensitizing agent coupled to theinterior material structure to produce ultrasound triggered cavitationcenters inside the exterior nanostructure. For example, the acousticsensitizing agent can include a fluorocarbon nanoemulsion. For example,when the device is deployed in a living organism, the exteriornanostructure of the device can be caused to rupture based on an appliedacoustic pulse (e.g., ultrasound pulse) to release the biologicalsubstance within the living organism.

Implementations of the exemplary device can include one or more of thefollowing exemplary features. In some implementations, for example, thedevice further can include an external coating formed of polyethyleneglycol (PEG) on the outer surface of the exterior nanostructure, inwhich the external coating is capable of preventing an immune systemresponse within a living organism when the device is deployed. In someimplementations, for example, the device further can include a targetingligand formed on the outer surface of the exterior nanostructure, inwhich the targeting ligand is capable of selectively binding to aparticular region of a cell or tissue of a living organism when thedevice is deployed. In some implementations, for example, the devicefurther can include an environmental sensing moiety formed on the outersurface of the exterior nanostructure, in which the environmentalsensing moiety is capable of chemically changing form to release thebiological substance based on at least one of a redox reaction, hypoxicreaction, or acidic pH in a local environment to which the device isdeployed.

In one example, a method for encapsulating a biological substanceincludes forming a biocompatible material onto a biological structure toform a coating structure enclosing the biological structure, the coatingstructure having a size in the nanometer range, in which the biologicalstructure preserves biological activity within the coating structure.

Implementations of the exemplary method can include one or more of thefollowing exemplary features. In some implementations, for example, themethod can further include forming the biological structure comprisingby cross-reacting a biological substance with a poly-cationic polymermaterial to form a positively-charged surface in the plurality ofregions of the biological structure. For example, the biologicalsubstance can include a virus, bacteria, protein, enzyme, prodrug,and/or nucleic acid vector including a DNA or an RNA. For example, thebiocompatible material can include silica. For example, thepoly-cationic polymer material can include poly-l-lysine. In someimplementations, for example, the forming the coating structure caninclude a charge-mediated silica sol-gel condensation reaction directlyonto the surface of the biological structure, in which the formedcoating structure includes an enveloping silica matrix encapsulating thebiological structure. In some implementations, for example, the methodcan further include attaching a sensitizing agent to the biologicalstructure, prior to the forming the biological material onto thebiological structure, to produce ultrasound triggered cavitation centersinside the coating structure. For example, the sensitizing agent caninclude a fluorocarbon nanoemulsion.

Further Examples

The following examples are illustrative of several embodiments of thepresent technology. Other exemplary embodiments of the presenttechnology may be presented prior to the following listed examples, orafter the following listed examples.

In one example of the present technology (example 1), a method ofencapsulating a biological entity includes templating a biocompatiblematerial onto a biological structure to form a coating structureenclosing the biological structure, the coating structure having a sizein the nanometer range, in which the coated biological structurepreserves its biological activity within the coating structure.

In another example of the present technology (example 2), a method ofencapsulating a biological entity includes using a biocompatiblematerial to form outside a biological structure of a dimension in ananometer range as a coating structure to enclose the biologicalstructure and to preserve biological activity of the biologicalstructure.

Example 3 includes the method of example 1 or 2, in which the biologicalstructure includes a virus.

Example 4 includes the method of example 1 or 2, in which thebiocompatible material includes silica.

Example 5 includes the method of example 1 or 2, which further includesusing an external triggering mechanism to release the biologicalstructure from the coating structure.

Example 6 includes the method of example 1 or 2, which further includesusing a targeting moiety incorporated in the coating structure torelease the biological structure from the coating structure.

Example 7 includes the method of example 1 or 2, which further includesaltering the surface charge on the biological structure, the alteringincluding cross-reacting the biological structure with a poly-cationicsubstrate to form a positively charged surface compatible with thetemplating process.

Example 8 includes the method of example 7, in which the poly-cationicsubstrate includes poly-l-lysine.

Example 9 includes the method of example 1 or 2, which further includesimplementing a charge-mediated silica sol-gel condensation reactiondirectly onto the surface of the biological structure to form anenveloping silica matrix forming the coating structure around thebiological structure.

Example 10 includes the method of example 1 or 2, which further includesadding a sensitizing agent comprising a nanoemulsion to form ultrasoundtriggered cavitation centers in the coating structure, in which theadding the sensitizing agent is implemented prior to the templating.

Example 11 includes the method of example 10, in which the sensitizingagent includes fluorocarbon emulsions.

Example 12 includes the method of example 1 or 2, in which thetemplating includes including one or more of a drug or a therapeuticagent with the biological structure to be enclosed in the coatingstructure.

Example 13 includes the method of example 1 or 2, in which thetemplating includes adding an iron oxide constituent or other nanoscalematerial with the biocompatible material to form the coating structure,the iron oxide constituent or the other nanoscale material providing anagent to enhance imaging of the coating structure.

Example 14 includes the method of example 1 or 2, which further includesfunctionalizing the surface of the coating structure.

Example 15 includes the method of example 14, in which thefunctionalizing includes adding polyethylene glycol (PEG) using silanechemistry.

While this patent document contains many specifics, these should not beconstrued as limitations on the scope of any invention or of what may beclaimed, but rather as descriptions of features that may be specific toparticular embodiments of particular inventions. Certain features thatare described in this patent document in the context of separateembodiments can also be implemented in combination in a singleembodiment. Conversely, various features that are described in thecontext of a single embodiment can also be implemented in multipleembodiments separately or in any suitable subcombination. Moreover,although features may be described above as acting in certaincombinations and even initially claimed as such, one or more featuresfrom a claimed combination can in some cases be excised from thecombination, and the claimed combination may be directed to asubcombination or variation of a subcombination.

Similarly, while operations are depicted in the drawings in a particularorder, this should not be understood as requiring that such operationsbe performed in the particular order shown or in sequential order, orthat all illustrated operations be performed, to achieve desirableresults. Moreover, the separation of various system components in theembodiments described in this patent document should not be understoodas requiring such separation in all embodiments.

Only a few implementations and examples are described and otherimplementations, enhancements and variations can be made based on whatis described and illustrated in this patent document.

What is claimed is:
 1. A method to produce a bioactive payload deliverydevice, comprising: forming an intermediate structure by binding apolymer material with a biological substance based on an electrostaticforce, wherein the formed intermediate structure includes a plurality ofregions presenting a net surface charge; and forming a coating structureof a biocompatible material directly on the formed intermediatestructure to enclose the biological substance, wherein the coatingstructure preserves biological activity of the biological substanceenclosed therein, thereby producing a bioactive payload delivery device.2. The method of claim 1, wherein the coating structure includes ananoparticle sized in a nanometer regime.
 3. The method of claim 1,wherein the biological substance includes at least one of a virus,bacteria, protein, enzyme, prodrug, or nucleic acid vector including aDNA or an RNA.
 4. The method of claim 1, wherein the biocompatiblematerial includes silica.
 5. The method of claim 1, wherein the formingthe intermediate structure includes cross-reacting the biologicalsubstance with a poly-cationic polymer material to form apositively-charged surface in the plurality of regions of theintermediate structure.
 6. The method of claim 5, wherein thepoly-cationic polymer material includes poly-l-lysine.
 7. The method ofclaim 6, wherein the forming the coating structure includes acharge-mediated silica sol-gel condensation reaction directly onto thesurface of the intermediate structure, wherein the formed coatingstructure includes an enveloping silica matrix encapsulating thebiological substance.
 8. The method of claim 1, further comprising:adding a sensitizing agent to produce ultrasound triggered cavitationcenters inside the coating structure, wherein the adding the sensitizingagent is implemented prior to the forming the coating structure.
 9. Themethod of claim 8, wherein the sensitizing agent includes a fluorocarbonnanoemulsion.
 10. The method of claim 8, further comprising deliveringthe biological substance to a target cell or tissue in a livingorganism, the delivering comprising: injecting the produced bioactivepayload delivery device through vasculature of the body, wherein thebioactive payload delivery device extravasates from the vasculature tothe target cell or tissue; and applying acoustic energy to an area ofthe living organism including or proximate to the target cell or tissueto cause the sensitizing agent to rupture the coating structure andrelease the biological substance to the target cell or tissue.
 11. Themethod of claim 1, further comprising: adding a pharmaceutical drug or atherapeutic agent with the intermediate structure to be enclosed in thecoating structure, wherein the adding the pharmaceutical drug or thetherapeutic agent is implemented prior to the forming the coatingstructure.
 12. The method of claim 1, further comprising: adding an ironoxide constituent or other nanoscale material with the biocompatiblematerial to form the coating structure, wherein the added iron oxideconstituent or the other nanoscale material provides an agent to enhanceimaging of the coating structure.
 13. The method of claim 1, wherein thecoating structure stabilizes the biological substance enclosed therein,thereby allowing thermal stability at temperatures including 80° C. to−37° C.
 14. The method of claim 1, further comprising: lyophilizing orcritical point drying the produced bioactive payload delivery device,wherein the coating structure preserves biological activity of thebiological substance enclosed within the lyophilized or critical pointdried bioactive payload delivery device.
 15. The method of claim 1,further comprising: functionalizing an exterior surface of the coatingstructure.
 16. The method of claim 15, wherein the functionalizingincludes adding polyethylene glycol (PEG) to provide a secondary coatingcapable of preventing an immune system response within a living organismin which the bioactive payload delivery device is deployed.
 17. Themethod of claim 15, wherein the functionalizing includes attaching atargeting ligand capable of selectively binding to a particular regionof a cell or tissue of a living organism in which the bioactive payloaddelivery device is deployed.
 18. The method of claim 15, wherein thefunctionalizing includes attaching an environmental sensing moiety tothe external surface, the environmental sensing moiety capable ofchemically changing form based on at least one of a redox reaction,hypoxic reaction, or acidic pH in a local environment to which thebioactive payload delivery device is deployed.
 19. The method of claim1, further comprising: prior to the forming the coating structure,binding a targeting moiety to the intermediate structure to enablecontrolled release of the biological substance from the coatingstructure.
 20. The method of claim 19, further comprising: deploying theproduced bioactive payload delivery device in a fluidic media to atarget substance; and applying an external triggering signal to an areaincluding or proximate to the target substance to cause the targetingmoiety to release the biological substance from the coating structure tothe target substance.
 21. A bioactive payload delivery device,comprising: an interior material structure including a polymer materialand a biological substance that are bound to each other via anelectrostatic interaction, wherein the interior material structureincludes a plurality of regions presenting a net surface charge; and anexterior nanostructure formed of a biocompatible material to encapsulatethe interior structured material, thus preserving biological activity ofthe encapsulated biological substance.
 22. The device of claim 21,wherein the exterior nanostructure protects the biological substancefrom degradation from external environmental factors including pH,temperature, pressure, and chemical substances in an environment wherethe bioactive payload delivery device is deployed.
 23. The device ofclaim 21, wherein an outer surface of the exterior nanostructure isfunctionalized with a tumor targeting ligand to cause the bioactivepayload delivery device to selectively accumulate in a tumor region overother tissues.
 24. The device of claim 21, wherein an outer surface ofthe exterior nanostructure is functionalized with an agent to increasecirculation time by reducing uptake from undesired body tissues, organs,and systems, the agent including at least one of polyethylene glycol, azwitterionic compound, or a patient-specific coating such as cellmembranes.
 25. The device of claim 21, wherein the biological substanceincludes at least one of a virus, bacteria, protein, enzyme, prodrug, ornucleic acid vector including a DNA or an RNA.
 26. The device of claim21, wherein the biocompatible material includes silica.
 27. The deviceof claim 21, further comprising: an acoustic sensitizing agent coupledto the interior material structure to produce ultrasound triggeredcavitation centers inside the exterior nanostructure.
 28. The device ofclaim 27, wherein the acoustic sensitizing agent includes a fluorocarbonnanoemulsion.
 29. The device of claim 27, wherein, when deployed in aliving organism, the exterior nanostructure of the device ruptures basedon an applied acoustic pulse to release the biological substance withinthe living organism.
 30. The device of claim 21, further comprising: anexternal coating formed of polyethylene glycol (PEG) on the outersurface of the exterior nanostructure, the external coating capable ofpreventing an immune system response within a living organism when thedevice is deployed.
 31. The device of claim 21, further comprising: atargeting ligand formed on the outer surface of the exteriornanostructure, the targeting ligand capable of selectively binding to aparticular region of a cell or tissue of a living organism when thedevice is deployed.
 32. The device of claim 21, further comprising: anenvironmental sensing moiety formed on the outer surface of the exteriornanostructure, the environmental sensing moiety capable of chemicallychanging form to release the biological substance based on at least oneof a redox reaction, hypoxic reaction, or acidic pH in a localenvironment to which the device is deployed.
 33. A method forencapsulating a biological substance, comprising: forming abiocompatible material onto a biological structure to form a coatingstructure enclosing the biological structure, the coating structurehaving a size in the nanometer range, wherein the biological structurepreserves biological activity within the coating structure.
 34. Themethod of claim 33, wherein the biocompatible material includes silica.35. The method of claim 33, further comprising: forming the biologicalstructure comprising by cross-reacting a biological substance with apoly-cationic polymer material to form a positively-charged surface inthe plurality of regions of the biological structure.
 36. The method ofclaim 35, wherein the biological substance includes at least one of avirus, bacteria, protein, enzyme, prodrug, or nucleic acid vectorincluding a DNA or an RNA.
 37. The method of claim 35, wherein thepoly-cationic polymer material includes poly-l-lysine.
 38. The method ofclaim 35, wherein the forming the coating structure includes acharge-mediated silica sol-gel condensation reaction directly onto thesurface of the biological structure, wherein the formed coatingstructure includes an enveloping silica matrix encapsulating thebiological structure.
 39. The method of claim 33, further comprising:attaching a sensitizing agent to the biological structure, prior to theforming the biological material onto the biological structure, toproduce ultrasound triggered cavitation centers inside the coatingstructure.
 40. The method of claim 39, wherein the sensitizing agentincludes a fluorocarbon nanoemulsion.